Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne bacterial pathogen that is able to escape host immune defenses to establish chronic infections in the skin, heart, joints, and central nervous system. Despite significant advances in the understanding of B. burgdorferi pathogenesis, studies to understand mechanisms of host immune evasion and other virulence properties of B. burgdorferi have been slowed by a paucity of genetic tools-particularly those amenable to high-throughput screening. Massively parallel sequencing is a rapidly developing technology for the study of bacterial pathogenesis. Dr. Andrew Camilli, a co-investigator on this proposal, has paired massively parallel sequencing with transposon mutagenesis in a strategy called Tn-seq. Tn-seq involves screening of a library against a selective pressure and then sequencing the flanking regions to the transposon en masse, to identify the relative frequency of the mutants before and after selection. Dr. Tao Lin's laboratory has developed the first transposon mutant library in B. burgdorferi. This proposal pairs the expertise of Dr. Lin's laboratory in transposon mutagenesis with Dr. Hu's laboratory's expertise in the use of Tn-seq and in immune responses to B. burgdorferi. In our preliminary studies, we injected mice with a mini-library of 10 transposon mutants at different inoculation amounts. We found that Tn-seq is robust and reproducible in identifying B. burgdorferi mutants from joint tissue. However, we found that there is variability between mice in the establishment of specific clones in a joint suggesting that there may be a significant bottleneck to survival at inoculation. However, while there was significant individual variation, averaging of the results over 5 mice resulted in a distribution of mutants that closely matched the input for most strains. In Aim 1, we propose to use Tn-seq to first determine the kinetics of dissemination of B. burgdorferi from the inoculation site and to characterize the extent of the bottleneck to survival from the initial inoculation. Little is currently known about he population dynamics of how Borrelia survive and disseminate to distal sites. These studies will provide important insight into these mechanisms on a population basis. We will also establish the contribution of adaptive and innate immunity to the bottleneck using SCID and MyD88-/- mice. These studies will allow us to optimize the study design for Aim 2 where we will screen the entire library in wild type, SCID and MyD88-/- mice using Tn-seq. Screening of the library will allow us to identify insertions into genes that result in a relative loss or gain of fitness for infection of the mouse and establishment of infection at specific distal sites. The development of Tn-seq for use in the study of an in vivo model of B. burgdorferi infection has the potential to greatly accelerate our understanding of genes involved in the pathogenesis of disease and the ability of the organism to evade our host immune defenses.